CN112921089A

CN112921089A - Composition and kit for methylation noninvasive early detection of gynecological tumors

Info

Publication number: CN112921089A
Application number: CN202110200262.2A
Authority: CN
Inventors: 刘沛; 王林海; 王紫芸
Original assignee: Beijing Originpoly Bio Tec Co Ltd
Current assignee: Beijing Originpoly Bio Tec Co Ltd
Priority date: 2021-02-23
Filing date: 2021-02-23
Publication date: 2021-06-08

Abstract

The invention discloses a composition and a kit for methylation noninvasive early detection of gynecological tumors, wherein the composition comprises a target gene detection primer for detecting a methylation site of a target gene, and the target gene comprises PTGER4, HAND2, PAX1, HOXA9, FOXD3, OPCML, HS3ST2 and JAM 3. The kit comprises detection primers and probes designed aiming at the genes of the composition, detection primers and probes of reference genes, PCR reaction liquid and other reagents. The detection of the methylation state of the gene can be used for carrying out non-invasive early screening on various gynecological tumors at one time, has the advantages of high detection speed, low cross contamination, final result accuracy guaranteed by a logistic regression algorithm and the like, and helps to realize the purposes of early discovery, early diagnosis and early treatment of malignant gynecological tumors.

Description

Composition and kit for methylation noninvasive early detection of gynecological tumors

Technical Field

The invention relates to the technical field of nucleic acid in-vitro diagnosis, in particular to application of a specific gene methylation marker in early detection and auxiliary diagnosis of gynecological tumors.

Background

Cervical cancer, endometrial cancer and ovarian cancer are the three most common malignant tumors in gynecological tumors in China. The incidence of cervical cancer in China is high, and the incidence of endometrial cancer is increased year by year in recent years. In terms of the survival rate of patients, the tumor cells of endometrial cancer are limited in the uterus and are not easy to spread, and the 5-year survival rate of early patients can reach 70-80 percent; in areas with better medical conditions, the 5-year survival rate for early cervical cancer can also reach about 80%. Although the incidence rate of ovarian cancer is not as high as that of other two gynecological tumors, the mortality rate is not as great, the prognosis of patients with advanced ovarian cancer is not good, and the data show that about 75% of patients with ovarian cancer in China are in advanced stage at the initial diagnosis, and the 5-year survival rate is less than 30%.

Cervical cancer is one of common gynecological malignant tumors, and the incidence rate of the cervical cancer is the second place among female malignant tumors in China and is behind breast cancer. According to worldwide statistics, there are about 50 ten thousand new cases of cervical cancer each year, accounting for 5% of all new cases of cancer, with more than 80% occurring in developing countries. Every year, 13 thousands of new cases of cervical cancer exist in China, and account for 28 percent of the total number of new cases of cervical cancer in the world. The peak age of the disease is 40-60 years, and a large number of researches show that the onset age of cervical cancer is in a trend of younger age. The incidence of cervical cancer is distributed with regional differences, rural areas are higher than cities, mountain areas are higher than plains, and developing countries are higher than developed countries. Therefore, there is a great need for the diagnosis and treatment of cervical cancer on a national scale. On the other hand, the occurrence of cervical cancer can be effectively controlled by the examination and management of precancerous lesions. Experience in western countries has shown that the incidence of cervical cancer is reduced by 70% to 90% in closely screened populations.

Early screening is an effective method for preventing cervical cancer precancerous lesion and cervical cancer, and the detection means commonly used in clinic at present mainly comprises liquid-based cytology detection and HPV detection. Compared with the traditional pap smear method (the sensitivity is between 30 and 87 percent, and sometimes the sensitivity is even lower than 20 percent), the liquid-based cytological detection has certain improvement, but a certain proportion of false negative results still exist (researches show that the false negative probability is about 50 percent). Since persistent high-risk HPV infection is the leading cause of cervical lesions, detection of HPV nucleotides has a very high sensitivity in detecting cervical lesions (about 90% in literature data), but since only a small fraction of high-risk HPV infected patients show cervical lesions, HPV detection shows very low specificity (about 60.7%), especially in young women. Therefore, a detection method with better accuracy and specificity is still lacked to be applied to clinical diagnosis.

Endometrial cancer is a group of epithelial malignant tumors occurring in the endometrium, also called endometrial cancer, and is one of three common malignant tumors of the female genital tract. It is most common in perimenopausal and postmenopausal women. With the increase in the average life span of the population and the change in lifestyle, the incidence of endometrial cancer is on a continuing rising and younger trend for nearly 20 years. In western countries, endometrial cancer already accounts for the first incidence of malignant tumors of the female reproductive system, and in China, the endometrial cancer accounts for about 20% -30% of the second common gynecological malignant tumor after cervical cancer. The incidence of endometrial cancer in some developed cities reaches the first gynecological malignancy.

Early screening is also an effective method for preventing endometrial cancer, and the detection means commonly used in clinic at present mainly comprise B-ultrasonic examination and hysteroscopy. The B-ultrasonic examination can know the size of the uterus, the thickness of the endometrium, whether echo is uneven or neoplasms exist in the uterine cavity, whether muscle layer infiltration exists and the degree thereof, and the diagnosis coincidence rate reaches more than 80 percent. Transvaginal ultrasound is more advantageous than transabdominal ultrasound because endometrial cancer patients are obese. Because of the convenience and non-invasiveness of the B-mode ultrasonography, the B-mode ultrasonography is the most routine examination for diagnosing endometrial cancer and is also a primary screening method. The existence of a cancer focus, the position, the size and the pathological change range of the cancer focus, the existence of the cervical canal and the affected part can be directly observed under the hysteroscope; the biopsy of suspicious lesion materials under direct vision is beneficial to finding out smaller or earlier lesion, and the missed diagnosis rate of endometrial cancer is reduced. The biopsy accuracy under the direct vision of the hysteroscope is close to 100%. Hysteroscopy and segmental curettage have complications such as hemorrhage, infection, perforation of uterus, cervical laceration, induced abortion complex reaction, etc., and hysteroscopy has risks such as water poisoning, etc. There is controversy as to whether hysteroscopy can lead to endometrial cancer dissemination, and most studies currently suggest that hysteroscopy does not affect the prognosis of endometrial cancer.

In China, the annual incidence of ovarian cancer is at the 3 rd position of female reproductive system tumors, and is located behind cervical cancer and uterine body malignant tumors, the ovarian cancer tends to rise year by year, and the mortality is located at the top of female reproductive tract malignant tumors, so the ovarian cancer is a malignant tumor seriously threatening the health of women. Ovarian malignancies encompass a variety of pathological types, the most common of which is epithelial cancer, accounting for about 70% of ovarian malignancies, followed by malignant germ cell tumors and sex cord stromal tumors, accounting for about 20% and 5% of each.

Currently, the main methods for monitoring the treatment and recurrence of ovarian cancer include imaging examination, such as transvaginal ultrasound (TVUS), tumor marker examination, such as CA125, HE4, histopathological examination, and the like. The transvaginal ultrasonography (TVS) probe is close to the ovary, has high image resolution, is not interfered by obesity and intestinal qi, and has higher sensitivity and specificity on the diagnosis of ovarian cancer. A woman with no sexual history may use transrectal ultrasound. Transabdominal ultrasound is an important complement to vaginal ultrasound, such as an excessively large tumor, where vaginal ultrasound does not achieve a view of the entire tumor. In addition, the condition of invasion of the ovarian cancer to peripheral organs, retroperitoneal lymph node metastasis and abdominal cavity implantation metastasis can be evaluated by the abdominal ultrasound, and the condition of ureterectasia, ascites and peritoneal implantation exist. In the aspect of ultrasonic intervention, for a patient who is expected to have difficulty in satisfactorily reducing tumors or weak bodies and cannot tolerate large operations, ultrasonic guided puncture can be selected for obtaining cytological or pathological diagnosis. The puncture part can be selected from pelvic tumor, thickened omentum majus, peritoneum, etc. In addition, when the thickening of the pelvic floor peritoneum is obvious, the puncture biopsy can be performed under the guidance of vaginal or rectal ultrasound. However, it should be noted that for preoperative comprehensive image assessment of isolated ovarian tumors without definite metastasis, especially those with suspicious early stage ovarian cancer, a needle biopsy needs to be carefully selected because iatrogenic tumor dissemination due to needle puncture is avoided. As a common ovarian cancer tumor marker, particularly a preferred tumor marker of serous ovarian cancer, the positive rate of CA125 is related to the tumor stage and the histological type, and the positive rate of patients with advanced serous cancer is obviously higher than that of patients with early and non-serous cancer (the positive rate of early ovarian cancer is about 43.50-65.70%, and the positive rate of advanced ovarian cancer is about 84.10-92.40%). Research shows that the application value of CA125 in postmenopausal people is higher, and the sensitivity (79.1-90.7%) and specificity (79.1-89.8%) of the CA125 in diagnosing ovarian cancer in postmenopausal people are superior to those in premenopausal people (the sensitivity is 69.8-87.5%, and the specificity is 63.3-85.7%). After surgery or chemotherapy, the correlation between blood CA125 concentration and disease progression is better in 87% to 94% of ovarian cancer cases, suggesting tumor progression or regression. CA125 can drop below 75% of the initial levels within 7 days after satisfactory tumor reduction. Most ovarian malignancies incorporate ascites or pleural effusion, and cancer cells can be found by cytological examination of ascites or pleural effusion. Histopathology is the gold standard for diagnosis. For patients with highly suspicious advanced ovarian cancer in clinic, the laparoscope exploration biopsy can not only obtain tissue specimens, but also observe the condition of tumor metastasis and distribution in the abdominal cavity and the pelvic cavity, and evaluate whether satisfactory tumor reduction surgery can be realized.

Non-invasive and low cost are desirable features for early screening of cancer. The detection methods for the malignant gynecological tumors are clinically conventional, but the detection methods have low detection rate and unsatisfactory specificity. Currently, there is no clinically effective early screening method.

DNA methylation is a modification of epigenetics, and studies have shown that DNA methylation can affect gene expression and silencing in normal mammalian cells; meanwhile, in human tumor research, DNA methylation can generally cause CpG island changes in the promoter region of tumor suppressor genes. Hypermethylation or hypomethylation of the promoter region of tumor suppressor genes may lead to cell transformation, making DNA methylation status a potential marker for tumor detection.

DNA methylation occurs primarily in the promoter region of genes and is often closely associated with inactivation of expression of cancer suppressor genes. The current methods applied to gene methylation detection mainly include: methylation-specific PCR (MSP), Bisulfite Sequencing PCR (BSP), and High Resolution Melting curve method (HRM). Methylation-specific PCR relies primarily on the binding of primers to a target template for PCR amplification to detect methylation sites; the bisulfite sequencing method relies on sequencing primers to perform PCR amplification, and on the basis, subsequent sequencing is performed to realize detection of methylation sites; the high resolution dissolution curve method distinguishes between methylated and unmethylated cases primarily by the change in dissolution temperature due to changes in the CG content of the sample. Each method has respective characteristics, the BSP method has higher result accuracy, is easy to visually judge and read, but has lower sensitivity, more complicated operation and high cost; the HRM method has relatively low sensitivity and slightly complex result analysis; the PCR method has high detection sensitivity, relatively low requirement on samples, short detection time, low cost and easy interpretation of results.

With the intensive research on tumors, it is gradually found that there are certain limitations in the biopsy technique during the diagnosis and treatment of cancer. The main performance is as follows: tumors are heterogeneous; certain patients have difficulty obtaining tissue for various reasons; when receiving a needle biopsy, there is also a risk of accelerating tumor metastasis; the hysteresis of tissue biopsy is also detrimental to patient treatment. There is therefore a greater demand for techniques for the diagnosis and detection of cancer.

The advent of liquid biopsy technology, which addresses the above-mentioned problems, has also advanced the time to diagnosis of cancer. Fluid biopsy is a technique, and more particularly a clinical solution. The liquid biopsy has the advantages of reducing the damage of biopsy by non-invasive sampling and effectively prolonging the life time of a patient with high cost performance. Relatively easily accessible to the tissue and non-invasive to the patient. However, the amount of circulating DNA in the plasma is low and is subject to degradation. Therefore, the difficulty of detecting the methylation of genes in the plasma is relatively high, the pretreatment process of a sample is paid attention to, the quality of bis-DNA is ensured, the design requirements on primers and probe sequences in an amplification reaction system are also very high, and the design of the primer and probe sequences is directly related to the final detection sensitivity and specificity of the whole detection means. Therefore, for the development of such detection kits, the sensitivity and specificity of the detection system need to be improved, and basically, the selection of target genes, the design of primers and probe sequences need to be focused except for the acquisition of samples.

Disclosure of Invention

The invention aims to solve the problems in the prior art, and provides a composition for early detection of multiple malignant gynecological tumors, which has high sensitivity and specificity, can obtain the occurrence risk of multiple malignant tumors through one-time detection, and has high accuracy, time saving and labor saving.

The detailed technical scheme of the invention is as follows:

the invention provides a composition for methylation noninvasive early detection of gynecological tumors, which comprises a target gene detection primer for detecting a methylation site of a target gene, wherein the target gene is PTGER4, HAND2, PAX1, HOXA9, FOXD3, OPCML, HS3ST2 and JAM 3.

The methylation degree of prostaglandin E receptor gene PTGER4 (prostagladin E receptor 4gene) is involved in the regulation of multiple gynecological tumors.

HAND2(Heart And Neural Crest variants Expressed 2) is a protein-encoding gene. Diseases related to HAND2 include cardiomyopathy and the like. Its associated pathways include cardiac development, NFAT and cardiac hypertrophy. The methylation process of HAND2 is involved in the development of tumors such as endometrial cancer, and is closely related to the tumor.

The PAX1(Pair box gene 1) gene is a member of the PAX gene family located on chromosome 20 and has a key role in fetal development and cell proliferation. Methylation of the PAX1 gene promoter plays an important epigenetic regulatory role in the development and progression of tumors. PAX1 is methylated and silenced in cervical and ovarian cancers, and thus PAX1 is also considered a tumor suppressor.

HOXA9(Homeobox a9) belongs to the HOX gene family, which encodes transcription factors that are critical for embryogenesis and are involved in canceration in a variety of different cancer types. In a recent study, we found that high HOXA9 promoter methylation was associated with poorer cancer-specific survival in patients with ovarian cancer and the like.

The FOXD3(Forkhead Box D3) gene, as a member of the Forkhead gene family, plays an important role in activities such as development, cell maintenance and the like, and simultaneously interacts with other transcription factors to influence the generation and development of tumors. Studies have shown FOXD3 to act as a tumor suppressor in a variety of different cancers. Yan et al found that FOXD3 inhibited tumor growth in non-small cell lung cancer; li et al disclose that FOXD3 is capable of inhibiting the development of colorectal cancer. In 2019, the research of Luo et al found that the methylation level of FOXD3 in ovarian cancer tissue samples is higher than that in normal ovarian tissue samples, and meanwhile, the expression of FOXD3 in ovarian cancer tissue samples is reduced; indicating that FOXD3 is expected to be a marker of human ovarian cancer.

The OPCML (Opioid binding protein/cell addition molecule-like) gene is a plasma membrane protein and acts as an Opioid receptor. Research shows that the OPCML gene has epigenetic inactivation condition in epithelial ovarian cancer, brain cancer, non-small cell cancer, bladder cancer, bile duct cancer, primary nasopharyngeal carcinoma, esophageal cancer, gastric cancer, liver cancer, colorectal cancer, mammary gland and cervical cancer, shows that OPCML has tumor inhibiting effect in many cancers, and the methylation and expression reduction of OPCML are considered to be associated with low survival rate. Meanwhile, the apparent silencing phenomenon of OPCML in human epithelial ovarian cancer is found to be more than 80%.

The HS3ST2 (heparin sulfate glucosamine 3sulfotransferase 2) gene encodes heparan sulfate 3-O-thiotransferase 2 enzyme, which is a key component involved in various biological activities in fine structure biosynthesis of Heparan Sulfate (HS). Each enzyme in this cascade has a tissue-specific role and serves as a substrate for subsequent reactions. Thus, even a single enzymatic change, including heparan sulfate 3-O-sulfur transferase 2, results in different HS structures being involved in several types of cancer.

JAM3(junctional addition molecule 3) is a member of the JAM gene family, which directly influences the tight junction between epithelial cells and endothelial cells. A number of literature studies report that JAM3, also commonly referred to as JAMC, is a ligation-modulating factor. In recent years, JAM3 has been reported to have important regulatory effects on tumors in tumor development, such as the manifestation of methylation in both colorectal and cervical cancers.

Although many related marker genes are found in the research of malignant gynecological tumors, in the past research, the correlation between the marker genes is not found, the conclusion about the correlation with diseases is mostly drawn independently, and the correlation is different, so that the correlation is not enough for clinical diagnosis. After a large amount of systematic researches, the inventor discovers that although each of the 8 genes is related to various cancers, the DNA methylation levels of the 8 genes are obviously higher in gynecological malignant tumors such as cervical cancer, endometrial cancer and ovarian cancer than in normal cells or gynecological benign tumors, and the methylation states of the genes are complementary in gynecological tumor patients with different histopathological types, so that the detection of the gynecological tumor patients can be maximally covered, and the detection can be used for assisting the early diagnosis of the gynecological tumors.

By designing corresponding primers or adding probes to the methylation sites of the 8 target genes and detecting the methylation regions of PTGER4, HAND2, PAX1, HOXA9, FOXD3, OPCML, HS3ST2 and JAM3 genes in free DNA in blood of a patient, the methylation state results of all the genes can be combined with each other, the purpose of early diagnosis is realized, and the occurrence risk of multiple gynecological malignant tumors can be obtained simultaneously by one-time detection.

Preferably, in the above composition, the nucleotide sequence of the target gene detection primer is as follows:

PTGER 4gene detection primers: 1-2 of SEQ ID NO;

HAND2 gene detection primer: 3-4 of SEQ ID NO;

PAX1 gene detection primers: 5-6 of SEQ ID NO;

HOXA9 gene detection primers: 7-8 of SEQ ID NO;

FOXD3 gene detection primers: 9-10 of SEQ ID NO;

OPCML gene detection primers: 11-12 of SEQ ID NO;

HS3ST2 gene detection primers: 13-14 of SEQ ID NO;

JAM3 gene detection primers: 15-16 of SEQ ID NO.

The target gene detection primers are non-methylation specific primers after target gene transformation, and can detect the methylation state of the target gene. In the primers, CG sites which are distributed less in the forward and reverse primers are even not contained, so that the converted sequence can be captured accurately, the combination with a template sequence before conversion is reduced, the detection specificity is improved, a foundation is provided for methylation region detection of a probe later, and the detection sensitivity of the probe is enhanced.

The amplification of the primer genes does not interfere with each other, the amplification efficiency of a plurality of genes in a detection system is consistent with the single amplification efficiency thereof, that is, the amplification efficiency of the genes in the multiple reaction system is not inhibited by the primers, so that the detection system can be set as a tube to detect five genes including four target genes and an internal reference gene, and at the moment, the five genes need to be represented in the form of Ct values through different fluorescence channels.

The primers are combined with clinical samples of accurate pathological information to determine a reasonable positive judgment value through an ROC curve, so that the screening accuracy of a reaction system and the early detection reliability of the gynecological tumor can be improved, the occurrence of false positive and false negative results can be avoided to the maximum extent, and the detection performance is integrally improved.

Preferably, the composition further comprises a target gene detection probe, and the specific nucleotide sequence is as follows:

PTGER 4gene detection probe: 17 in SEQ ID NO;

HAND2 gene detection probe: 18 in SEQ ID NO;

PAX1 gene detection probe: 19 in SEQ ID NO;

HOXA9 gene detection probes: 20 in SEQ ID NO;

FOXD3 gene detection probes: 21, SEQ ID NO;

detection probe for OPCML gene: 22 is SEQ ID NO;

HS3ST2 gene detection probe: 23, SEQ ID NO;

JAM3 gene detection probe: 24 is SEQ ID NO;

the 5 'end of the detection probe of the target gene is fluorescently labeled, the 3' end is labeled with MGB, and different target genes in the same detection system are labeled with different fluorophores.

The target gene detection probes are MGB probes, and the difference between the binding free energy delta G of the probes and the methylated templates and the binding free energy delta G of the probes and the unmethylated templates is 20kcal mol^-1And the MGB modification is introduced into the probe, so that the sequence of the probe is shortened, the recognition of the probe to the template sequence is greatly improved, the amplification of a methylation site is facilitated, and the specificity and the sensitivity of the detection are further improved.

Preferably, in the above composition, the specific positions of the fluorescent label and the MGB are as follows:

PTGER 4gene detection probe: marking FAM at the 5 'end of the sequence and marking MGB at the 3' end;

HAND2 gene detection probe: marking ROX at the 5 'end of the sequence and marking MGB at the 3' end;

PAX1 gene detection probe: the sequence is marked with CY5 at the 5 'end and MGB at the 3' end;

HOXA9 gene detection probes: HEX is marked at the 5 'end of the sequence, and MGB is marked at the 3' end of the sequence;

FOXD3 gene detection probes: marking FAM at the 5 'end of the sequence and marking MGB at the 3' end;

detection probe for OPCML gene: the sequence is marked with ROX at the 5 'end and MGB at the 3' end.

HS3ST2 gene detection probe: the sequence is marked with CY5 at the 5 'end and MGB at the 3' end;

JAM3 gene detection probe: the 5 'end of the sequence is marked with HEX, and the 3' end is marked with MGB.

The MGB probes marked by different fluorescence channels can be placed in a tube for reaction, so that the optimal amplification efficiency of 5 different target genes in a sample can be ensured, and the fluorescence curve is a standard S-shaped amplification curve and keeps consistent trend compared with the single amplification of each gene.

Preferably, the composition further comprises detection primers for reference genes GAPDH and beta-actin, and the specific nucleotide sequences are as follows:

GAPDH gene detection primer: 25 to 26 of the amino acid sequence shown in SEQ ID NO,

beta-actin gene detection primers: 27-28 of SEQ ID NO.

Preferably, the composition further comprises detection probes for reference genes GAPDH and beta-actin, and the specific nucleotide sequences are as follows:

GAPDH gene detection probe: 29, JOE marked at the 5 'end of the sequence and BHQ1 marked at the 3' end of the sequence;

beta-actin gene detection probe: 30, the 5 'end of the sequence is marked with JOE, and the 3' end is marked with BHQ 1.

The double internal reference genes are used as a control, and a specific primer probe is designed, so that the detection accuracy can be further increased, and the errors among detections can be reduced.

The invention also provides a methylation noninvasive early detection kit for gynecological tumors, which comprises any one of the compositions and PCR reaction liquid.

Preferably, in the kit, the PCR reaction solution comprises 0.5 to 1. mu.L of DNA Taq polymerase with a concentration of 1U/. mu.L, 2 to 5. mu.L of dNTPs with a concentration of 25mM, and Mg with a concentration of 1.5mM for each aliquot²⁺2-6. mu.L, 10 XDNA polymerase buffer 5. mu.L and purified water to make up 25. mu.L. Taq polymerase and dNTPs, Mg²⁺The ratio of 10 XDNA polymerase buffer is directly related to the amplification efficiency of the combination of primer and probe.

The PCR reaction system is specially aimed at bis-DNA amplification after bisulfite conversion, and comprises multiple primer probes, so that the selection of PCR reaction liquid is particularly important, the amplification efficiency of each gene primer probe in the system is similar to that of corresponding single amplification, the primers or probes in the system are ensured not to be mutually interfered, and the amplification effect of each group of primer probes is fully exerted. Different DNA Taq polymerases and the ratios of the different DNA Taq polymerases to other components need to be screened and verified, so that the optimal amplification efficiency of the whole multiplex amplification system is ensured.

The invention also provides a using method of the kit, which takes plasma as a sample, extracts free DNA, and carries out bisulfite conversion to obtain Bis-DNA; performing PCR amplification by using Bis-DNA as a template, wherein the PCR reaction conditions are as follows: pre-denaturation at 96 ℃ for 5 min; denaturation at 94 ℃ for 15s, annealing and extension at 60 ℃ for 35s, and 45 cycles; keeping at 25 deg.C for 10 min. Bisulfite conversion can convert unmethylated 5 'cytosines in DNA to uracil, while methylated 5' cytosines are unchanged, resulting in Bis-DNA. The transformation efficiency of the DNA and the final transformation yield of the Bis-DNA are ensured in the transformation process.

In the extraction of plasma free DNA, it is preferable to use a large-volume free nucleic acid extraction kit (DP710) by the magnetic bead method of Tiangen Biochemical technology (Beijing) Ltd while monitoring the DNA quality. The bisulfite conversion is preferably carried out using DNA bisulfite conversion kit (DP215) from Tiangen Biochemical technology (Beijing) Ltd.

Preferably, in the above method, after the PCR amplification, the positive judgment value is determined by using a logistic regression algorithm principle and an ROC curve, and a logistic regression algorithm is performed according to a difference Δ Ct value between the Ct value obtained by the target gene amplification and the average value of the Ct values obtained by the dual-reference gene amplification to obtain a P value, and if the P value is greater than 0.05, methylation occurs, that is, it is determined that the occurrence of the malignant gynecological tumor is high risk.

Compared with the prior art, the invention has the following beneficial effects:

the research of the invention finds that after PTGER4, HAND2, PAX1, HOXA9, FOXD3, OPCML, HS3ST2 and JAM3 are combined, methylation state detection is carried out on the genes, the results can be combined with each other to judge the occurrence risk of gynecological malignant tumors, the result accuracy is very high, the kit can be directly applied to clinical diagnosis, and the purpose of early predicting various malignant tumors by one-time detection is realized.

The optimal detection primer is designed aiming at the methylated sites of the genes PTGER4, HAND2, PAX1, HOXA9, FOXD3, OPCML, HS3ST2 and JAM3, and comprises not only a promoter region of the genes, but also a coding region of the genes. The detection composition detects possible patients with gynecological malignant tumor in advance by a molecular epigenetic method and a methylation detection technology, and prevents and treats the gynecological malignant tumor in advance.

The kit disclosed by the invention mainly adopts a multi-gene multi-channel fluorescence detection means, utilizes five fluorescent probe labels to accurately identify a methylated sequence through a specific primer probe and an optimized special methylated DNA Taq polymerase, and accurately detects the methylation sites of PTGER4, HAND2, PAX1, HOXA9, FOXD3, OPCML, HS3ST2 and JAM3 genes. Compared with a method for detecting only by using a PCR specific primer, the method can carry out double recognition on a methylated template sequence by adding a specific probe, and can obviously improve the sensitivity and the accuracy of detection. And meanwhile, double internal reference genes are introduced to be used as a control, so that the detection accuracy is further improved, and the errors among detections are reduced.

The invention adopts the technology to carry out methylation detection of multiple multi-channel fluorescent polygenes, the related detection samples are relatively easy to obtain, non-invasive, the detection method is simple to operate, the interpretation is intuitive, the result is obtained within 8 hours, the universal fluorescent quantitative PCR instrument can meet the detection requirement, the whole set of experimental process adopts a one-stop totally-enclosed form, the operation is simpler and more convenient, and the possibility of cross contamination is avoided.

The whole detection system of the invention comprises a primer probe, DNA polymerase and other design techniques, so that the invention has high detection sensitivity, better detection rate on a low-concentration template, mutual complementation of selected target genes, good specificity of the primer probe, high amplification efficiency and very sensitivity for detecting early gynecological malignant tumors. The high sensitivity of the detection of the kit is suitable for detecting early gynecological malignant tumors.

Drawings

FIG. 1 is an exemplary diagram of the PCR fluorescent detection results of the target gene and the reference gene (detection result of primer probe mixture 1);

FIG. 2 is an exemplary diagram of the PCR fluorescent detection results of the target gene and the reference gene (detection result of primer probe mixture 2);

FIG. 3 is a ROC curve obtained by detecting 300 samples with the methylation detection kit of example 1;

FIG. 4 is a ROC curve obtained by detecting 300 samples with the methylation detection kit in example 2.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments in order to make the technical field better understand the scheme of the present invention.

Example 1

Detection test of detection kit for gynecological malignant tumor related genes PTGER4, HAND2, PAX1, HOXA9, FOXD3, OPCML, HS3ST2 and JAM 3.

The specific nucleotide sequences of the primers and probes used are shown in the following table:

note: f represents a forward detection primer, R represents a reverse detection primer, and FP represents a detection probe. In this table, the probe sequences shown have been fluorescently labeled and quenched.

The kit components are as follows:

70 ovarian cancer samples with known and definite pathological information results were selected: 30 cases were identified as high-grade serous carcinoma, 20 cases of low-grade serous carcinoma, 10 cases of clear cell carcinoma, and 10 cases of mucinous carcinoma; 40 cases are benign samples of the ovary.

60 examples of endometrial cancer samples with known and definite pathological information results were selected: 15 cases were identified as endometrioid carcinoma, 15 cases of endometrial mucinous carcinoma, 15 cases of endometrial serous carcinoma, 15 cases of endometrial clear cell carcinoma; 40 cases are benign samples of the endometrium.

50 ovarian cancer samples with known and definite pathological information results were selected: 40 cases were identified as cervical squamous carcinoma and 10 cases were identified as cervical adenocarcinoma; 40 cases are benign samples of the cervix.

1. The 300 samples of gynecological benign and malignant tumor were extracted with free DNA in plasma using a large volume free nucleic acid extraction kit (DP710) by the magnetic bead method of Tiangen Biochemical technology (Beijing) Ltd, and the quality of DNA was monitored.

2. The extracted DNA is subjected to bisulfite conversion by using a DNA bisulfite conversion kit (DP215) of Tiangen Biochemical technology (Beijing) Co., Ltd., unmethylated cytosine (C) in the DNA is converted into uracil (U), and methylated cytosine (C) is not changed, so that converted bis-DNA is obtained. The conversion efficiency of the kit can reach 99 percent, which is higher than that of most bisulfite conversion kits in the market.

3. Preparing PCR reaction liquid and primer probe mixed liquid;

PCR reaction solution (25. mu.L/person)

Primer Probe Mixed solution 1 (10. mu.L/person)

Components	One part addition amount (mu L)
		PTGER4 gene-F (100. mu.M)	0.4
PTGER4 Gene-R (100. mu.M)	0.4
		PTGER4 gene-FP (100. mu.M)	0.1
HAND2 gene-F (100. mu.M)	0.35
		HAND2 gene-R (100. mu.M)	0.35
HAND2 gene-FP (100. mu.M)	0.15
		PAX1 gene-F (100. mu.M)	0.4
PAX1 gene-R (100. mu.M)	0.4
		PAX1 gene-FP (100. mu.M)	0.15
HOXA9 gene-F (100. mu.M)	0.3
		HOXA9 Gene-R (100. mu.M)	0.3
HOXA9 gene-FP (100. mu.M)	0.1
		GAPDH gene-F (100. mu.M)	0.05
GAPDH gene-R (100. mu.M)	0.05
		GAPDH gene-FP (100. mu.M)	0.05
Purified water	Make up to 5. mu.L

Primer probe mixed solution 2(10 mu L/person)

4. Sample adding: 20 mu L of negative and positive quality control products and the transformed Bis-DNA clinical sample are respectively added into the prepared system. Carrying out PCR reaction under the following conditions: pre-denaturation at 96 ℃ for 5 min; denaturation at 94 ℃ for 15s, annealing and extension at 60 ℃ for 35s, and 45 cycles; keeping at 25 deg.C for 10 min.

5. The amplification procedure was as follows:

step 1: pre-denaturation at 96 ℃ for 5 min;

step 2: denaturation at 94 ℃ for 15s, annealing and extension at 60 ℃ for 35s, and 45 cycles;

step3：25℃，10min；

signal Collection, FAM, HEX, ROX, Joe and CY5 signals were collected at 60 ℃.

6. Interpretation of results

(1) The internal standard channel has an S-shaped amplification curve, and the Ct value is less than or equal to 38, so that the result is effective;

(2) the Δ Ct values for each of the 8 genes are: Δ Ct (PTGER4) ═ Ct (PTGER4) -Ct (mean internal reference), Δ Ct (had 2) ═ Ct (had 2) -Ct (mean internal reference), Δ Ct (PAX1) ═ Ct (PAX1) -Ct (mean internal reference), Δ Ct (HOXA9) ═ Ct (HOXA9) -Ct (mean internal reference), Δ Ct (FOXD3) ═ Ct (FOXD3) -Ct (mean internal reference), Δ Ct (opcml) ((HOXA opcml) -Ct (mean internal reference), Δ Ct (HS3ST2) ═ Ct (HS3ST2) -Ct (mean internal reference), Δ Ct (JAM3) (ja) 3) -mean (java) internal reference), and Δ Ct (gapdh) (+ 2) (. beta.) -Ct.

(3) Determining a cut-off value P value by combining the delta Ct values of the 8 genes and applying a logistic regression algorithm, wherein when the P value is more than 0.05, the detection result is methylation; on the other hand, when the P value is 0.05 or less, the methylation is not detected.

7. Analysis of detection results

The reaction system of the kit is used for detecting 300 samples in total, wherein the samples comprise 70 ovarian cancer samples, 40 benign ovarian samples, 60 endometrial cancer samples, 40 benign endometrial samples, 50 cervical cancer samples and 40 benign cervical samples. The results are shown in the following table.

TABLE 300 test results of the samples

Comparing clinical pathological results, the combined ROC curve area obtained by using the methylation detection kit is 0.942 (see figure 2), the overall specificity is 95.0%, the ovarian cancer detection sensitivity is 92.8%, the endometrial cancer detection sensitivity is 90% and the cervical cancer detection sensitivity is 92%. Wherein the detection rates of high-grade serous ovarian cancer, low-grade serous ovarian cancer, clear cell ovarian cancer and mucinous ovarian cancer are 93.3%, 95%, 90% and 90% respectively; the detection rates for endometrioid carcinoma, endometrial mucinous carcinoma, endometrial serous carcinoma and endometrial clear cell carcinoma were 86.7%, 93.3% and 93.3%, respectively. The detection rate of cervical squamous carcinoma and cervical adenocarcinoma is 95% and 90%. The results show that the kit can better distinguish the good and malignant tumor samples of the gynecology.

Example 2

The other components in the kit are the same as in example 1.

Compared with clinical pathological results, the combined ROC curve area obtained by using the methylation detection kit is 0.894 (see figure 4), the overall specificity is 97.5%, the ovarian cancer detection sensitivity is 90%, the endometrial cancer detection sensitivity is 86.7%, and the cervical cancer detection sensitivity is 86%. Wherein the detection rates of high-grade serous ovarian cancer, low-grade serous ovarian cancer, clear cell ovarian cancer and mucinous ovarian cancer are respectively 90%, 90% and 90%; the detection rates for endometrioid carcinoma, endometrial mucinous carcinoma, endometrial serous carcinoma and endometrial clear cell carcinoma were 80%, 86.7% and 93.3%, respectively. The detection rate of cervical squamous carcinoma and cervical adenocarcinoma is 90% and 70%.

The inventive concept is explained in detail herein using specific examples, which are given only to aid in understanding the core concepts of the invention. It should be understood that any obvious modifications, equivalents and other improvements made by those skilled in the art without departing from the spirit of the present invention are included in the scope of the present invention.

SEQUENCE LISTING

<110> Beijing-originated Poa-gathering Biotech Co., Ltd

<120> composition and kit for methylation noninvasive early detection of gynecological tumors

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<170> PatentIn version 3.5

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Claims

1. The composition for methylation noninvasive early detection of gynecological tumors is characterized by comprising target gene detection primers for detecting methylation sites of target genes, wherein the target genes are PTGER4, HAND2, PAX1, HOXA9, FOXD3, OPCML, HS3ST2 and JAM 3.

2. The composition of claim 1, wherein the nucleotide sequence of the target gene detection primer is as follows:

PTGER 4gene detection primers: 1-2 of SEQ ID NO;

HAND2 gene detection primer: 3-4 of SEQ ID NO;

PAX1 gene detection primers: 5-6 of SEQ ID NO;

HOXA9 gene detection primers: 7-8 of SEQ ID NO;

FOXD3 gene detection primers: 9-10 of SEQ ID NO;

OPCML gene detection primers: 11-12 of SEQ ID NO;

HS3ST2 gene detection primers: 13-14 of SEQ ID NO;

JAM3 gene detection primers: 15-16 of SEQ ID NO.

3. The composition of claim 1, further comprising a target gene detection probe, wherein the specific nucleotide sequence is as follows:

PTGER 4gene detection probe: 17 in SEQ ID NO;

HAND2 gene detection probe: 18 in SEQ ID NO;

PAX1 gene detection probe: 19 in SEQ ID NO;

HOXA9 gene detection probes: 20 in SEQ ID NO;

FOXD3 gene detection probes: 21, SEQ ID NO;

detection probe for OPCML gene: 22 is SEQ ID NO;

HS3ST2 gene detection probe: 23, SEQ ID NO;

JAM3 gene detection probe: 24 is SEQ ID NO;

4. The composition of claim 3, wherein the specific labeling positions of the fluorescent label and the MGB are as follows:

5. The composition as claimed in claim 1, further comprising detection primers for reference genes GAPDH and β -actin, wherein the specific nucleotide sequences are as follows:

beta-actin gene detection primers: 27-28 of SEQ ID NO.

6. The composition as claimed in claim 5, further comprising detection probes for reference genes GAPDH and β -actin, wherein the specific nucleotide sequences are as follows:

7. A methylation noninvasive early gynecological tumor detection kit, which is characterized by comprising the composition of any one of claims 1-6 and a PCR reaction solution.

8. The kit according to claim 7, wherein the PCR reaction solution comprises 0.5 to 1. mu.L of DNA Taq polymerase at a concentration of 1U/. mu.L, 2 to 5. mu.L of dNTPs at a concentration of 25mM, and Mg at a concentration of 1.5mM for each aliquot²⁺2-6. mu.L, 10 XDNA polymerase buffer 5. mu.L and purified water to make up 25. mu.L.

9. The method of using the kit according to claim 7 or 8, wherein the plasma is used as a sample, free DNA is extracted, and bisulfite conversion is performed to obtain Bis-DNA; performing PCR amplification by using Bis-DNA as a template, wherein the PCR reaction conditions are as follows: pre-denaturation at 96 ℃ for 5 min; denaturation at 94 ℃ for 15s, annealing and extension at 60 ℃ for 35s, and 45 cycles; keeping at 25 deg.C for 10 min.

10. The use method of claim 9, wherein after the obtained PCR amplification, a positive judgment value is determined by using a logistic regression algorithm principle and an ROC curve, a logistic regression algorithm is calculated according to a difference value delta Ct value between a Ct value obtained by the target gene amplification and an average value of Ct values obtained by the double-reference gene amplification to obtain a P value, and if the P value is greater than 0.05, methylation occurs, so that the occurrence of the malignant gynecological tumor is determined to be high risk.